Journal: Research

Article Title: Hypoxic Reprogramming of ACOX1-Driven HSP90AB1 Crotonylation Stabilizes Thioredoxin to Orchestrate Redox Homeostasis in Oral Squamous Cell Carcinoma

doi: 10.34133/research.1129

Figure Lengend Snippet: Hypoxia induces the site-specific crotonylation of heat shock protein 90 alpha family class B member 1 (HSP90AB1) at lysine 265 (K265) to regulate redox homeostasis in OSCC. (A) Volcano plot of crotonylation sites in CAL27 cells after 24 h of hypoxia versus normoxia. Sites are colored red for fold changes >1.5 and P < 0.05 and blue for fold changes <−1.5 and P < 0.05. (B and C) Subcellular localization analysis of (B) up-regulated and (C) down-regulated crotonylated proteins. (D) Immunoprecipitation (IP) of endogenous HSP90AB1 in CAL27/HSC3 cells under normoxia/hypoxia (24 h) and probing with a pan-Kcr antibody (IgG, immunoglobulin G; WCL, whole-cell lysate). (E) IP of Flag-HSP90AB1 in HEK293T cells under normoxia and hypoxia probed with a pan-Kcr antibody. (F) Crotonylation levels of Flag-HSP90AB1 in HEK293T cells treated with sodium crotonate (0 to 10 mM) under normoxia (24 h). (G) Domain structure of HSP90AB1 (NTD, N-terminal domain; MD, middle domain; CD, charged domain; CTD, C-terminal domain) and K265 mutation schematic (K, lysine; R, arginine; Q, glutamine). (H and I) IP of Flag-HSP90AB1 in HEK293T cells reconstituted with HSP90AB1 variants (wild type [WT], K265R, and K265Q) under normoxia/hypoxia (24 h) and probed with (H) pan-Kcr and (I) site-specific K265cr antibodies. (J) Representative confocal images of DCFH-DA staining in HSP90AB1-knockdown CAL27/HSC3 cells reconstituted with HSP90AB1 variants under normoxia/hypoxia (24 h) (200×, scale bars, 100 μm). NC, negative control; N, normoxia; H, hypoxia. (K) Quantification of DCFH-DA fluorescence intensity in HSP90AB1-knockdown CAL27/HSC3 cells reconstituted with HSP90AB1 variants under normoxia/hypoxia (24 h) (** P < 0.01 and *** P < 0.001, one-way ANOVA).

Article Snippet: The His-tagged HSP90AB1 fusion protein and His peptide were purchased from Cusabio Technology Co., Ltd. (Wuhan, China) and Yeasen Biotechnology Co., Ltd. (Shanghai, China).

Techniques: Immunoprecipitation, Mutagenesis, Staining, Knockdown, Negative Control, Fluorescence

Journal: Research

Article Title: Hypoxic Reprogramming of ACOX1-Driven HSP90AB1 Crotonylation Stabilizes Thioredoxin to Orchestrate Redox Homeostasis in Oral Squamous Cell Carcinoma

doi: 10.34133/research.1129

Figure Lengend Snippet: Experimental analysis of the HSP90AB1–TXN interaction and functional relationships. (A) Venn diagram comparing the hypoxia-up-regulated proteome (758 proteins, green) and the HSP90AB1 interactome (843 proteins, blue) identified by co-IP/mass spectrometry (MS), showing 98 overlapping candidates. (B) Protein–protein interaction network of the 98 candidates from panel (A). (C) Structural models of the HSP90AB1–TXN complex: (bottom) docked conformation (HSP90AB1, green; TXN, purple); (top) 3-dimensional interaction diagram of interface residues (HSP90AB1, green; TXN, cyan). (D) Endogenous co-IP of HSP90AB1 and TXN in CAL27/HSC3 cells. IgG: negative control. (E) In vitro pull-down assay of His-HSP90AB1 with TXN. (F) Immunofluorescence (IF) staining of HSP90AB1 (red) and TXN (green) in CAL27/HSC3 cells. Nuclei counterstained with DAPI (blue) (600×, scale bars, 20 μm). (G) Western blot analysis of the indicated proteins in CAL27/HSC3 cells treated with NVP-BEP800 (0 to 2 μM, 24 h). (H) Co-IP of HSP90AB1 and TXN in CAL27/HSC3 cells following NVP-BEP800 treatment (2 μM). (I) Representative confocal microscopy images of DCFH-DA staining in CAL27/HSC3 cells treated with NVP-BEP800 (0 to 2 μM, 24 h) (200×, scale bars, 100 μm). (J) Representative confocal microscopy images of DCFH-DA-stained CAL27/HSC3 cells with or without TXN overexpression and NVP-BEP800 treatment (200×, scale bars, 100 μm). ovTXN, TXN overexpression. (K) Quantification of DCFH-DA fluorescence intensity in CAL27/HSC3 cells treated with NVP-BEP800 (0 to 2 μM, 24 h) (** P < 0.01 and *** P < 0.001, one-way ANOVA). (L) Quantification of DCFH-DA fluorescence intensity in CAL27/HSC3 cells with or without TXN overexpression and NVP-BEP800 treatment (2 μM) (*** P < 0.001, one-way ANOVA).

Article Snippet: The His-tagged HSP90AB1 fusion protein and His peptide were purchased from Cusabio Technology Co., Ltd. (Wuhan, China) and Yeasen Biotechnology Co., Ltd. (Shanghai, China).

Techniques: Functional Assay, Co-Immunoprecipitation Assay, Mass Spectrometry, Negative Control, In Vitro, Pull Down Assay, Immunofluorescence, Staining, Western Blot, Confocal Microscopy, Over Expression, Fluorescence

Journal: Research

Article Title: Hypoxic Reprogramming of ACOX1-Driven HSP90AB1 Crotonylation Stabilizes Thioredoxin to Orchestrate Redox Homeostasis in Oral Squamous Cell Carcinoma

doi: 10.34133/research.1129

Figure Lengend Snippet: K265 crotonylation induces conformational remodeling of HSP90AB1 to enhance TXN binding and stabilization. (A) Co-IP of endogenous HSP90AB1–TXN complexes in CAL27/HSC3 cells under normoxia or hypoxia. (B) Molecular dynamics simulation snapshots (100 ns) of the HSP90AB1–TXN complex: HSP90AB1 with K265 crotonylation (red), TXN (blue), and the crotonyl moiety at K265 (green). (C) Root mean square deviation (RMSD) trajectory of the HSP90AB1–TXN complex during molecular dynamics simulation. (D) Root mean square fluctuation (RMSF) per residue analysis of HSP90AB1 during simulation. (E) Radius of gyration measurements of HSP90AB1 during simulation. (F) Secondary structure composition analysis of HSP90AB1 (Define Secondary Structure of Proteins [DSSP] method). (G) Hydrogen bond formation between HSP90AB1 and TXN with simulation time. (H) Solvent-accessible surface area (SASA) measurements of the HSP90AB1–TXN complex during simulation. (I) Structural superposition of the initial (red) and equilibrated (cyan) HSP90AB1 conformations. (J) Molecular docking model of K265cr-modified HSP90AB1 (green) with TXN (purple/cyan). (K) Co-IP of Flag-HSP90AB1 in HSP90AB1-knockdown CAL27/HSC3 cells reconstituted with HSP90AB1 variants under normoxia/hypoxia (24 h). (L) Representative confocal microscopy images of DCFH-DA-stained CAL27/HSC3 cells expressing HSP90AB1 variants with/without TXN overexpression under normoxia/hypoxia (24 h) (200×, scale bars, 100 μm). (M) Quantification of DCFH-DA fluorescence intensity in CAL27/HSC3 cells expressing HSP90AB1 variants with/without TXN overexpression under normoxia/hypoxia (24 h) (** P < 0.01 and *** P < 0.001, one-way ANOVA).

Article Snippet: The His-tagged HSP90AB1 fusion protein and His peptide were purchased from Cusabio Technology Co., Ltd. (Wuhan, China) and Yeasen Biotechnology Co., Ltd. (Shanghai, China).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Residue, Solvent, Modification, Knockdown, Confocal Microscopy, Staining, Expressing, Over Expression, Fluorescence

Journal: Research

Article Title: Hypoxic Reprogramming of ACOX1-Driven HSP90AB1 Crotonylation Stabilizes Thioredoxin to Orchestrate Redox Homeostasis in Oral Squamous Cell Carcinoma

doi: 10.34133/research.1129

Figure Lengend Snippet: HIF-1α transcriptionally activates acyl-CoA oxidase 1 (ACOX1) to promote HSP90AB1 K265 crotonylation. (A) Schematic diagram of the crotonyl-CoA biosynthesis pathway. (B) Scatter plot of HIF-1α versus ACOX1 mRNA expression levels in OSCC samples from the TCGA cohort ( n = 338, Pearson correlation). (C) qPCR analysis of ACOX1 mRNA levels in OSCC cells under normoxia and hypoxia (24 h) (* P < 0.05 and ** P < 0.01, unpaired t test). (D) Western blot analysis of the ACOX1 protein in OSCC cells under normoxia and hypoxia (24 h). β-Actin: loading control. (E) qPCR analysis of ACOX1 mRNA levels in shRNA-transduced CAL27/HSC3 cells (** P < 0.01 and *** P < 0.001, one-way ANOVA). (F) Western blot analysis of HSP90AB1 K265cr in scramble control versus ACOX1 -knockdown CAL27/HSC3 cells under normoxia/hypoxia (24 h). (G) Western blot analysis of HSP90AB1 K265cr in ACOX1 -knockdown CAL27/HSC3 cells treated with crotonyl-CoA (50 μM) or butyryl-CoA (50 μM). (H) qPCR analysis of ACOX1 mRNA levels in CAL27/HSC3 cells overexpressing ACOX1 (*** P < 0.001, one-way ANOVA). (I) Western blot confirmation of ACOX1 protein levels in the overexpression system. (J) Western blot analysis of HSP90AB1 K265cr in CAL27/HSC3 cells overexpressing ACOX1 under normoxia and hypoxia (24 h). (K) Diagram of the predicted HIF-1α-binding sites in the ACOX1 promoter region (JASPAR database). (L) Dual-luciferase reporter activity of the ACOX1-WT promoter and mutant (ACOX1-MUT) under normoxia/hypoxia (** P < 0.01 and *** P < 0.001, one-way ANOVA). (M) Agarose gel electrophoresis of Cleavage Under Targets and Tagmentation (CUT&Tag) products using an anti-HIF-1α antibody. (N) CUT&Tag–qPCR analysis of the association of HIF-1α with the ACOX1 promoter (*** P < 0.001, unpaired t test). (O) Western blot analysis of ACOX1 and HSP90AB1 K265cr in CAL27/HSC3 cells treated with dimethyloxalylglycine (DMOG; 2 mM) or CAY10585 (CAY; 10 μM) under normoxia/hypoxia (24 h).

Article Snippet: The His-tagged HSP90AB1 fusion protein and His peptide were purchased from Cusabio Technology Co., Ltd. (Wuhan, China) and Yeasen Biotechnology Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot, Control, shRNA, Knockdown, Over Expression, Binding Assay, Luciferase, Activity Assay, Mutagenesis, Agarose Gel Electrophoresis

Journal: Research

Article Title: Hypoxic Reprogramming of ACOX1-Driven HSP90AB1 Crotonylation Stabilizes Thioredoxin to Orchestrate Redox Homeostasis in Oral Squamous Cell Carcinoma

doi: 10.34133/research.1129

Figure Lengend Snippet: Clinical and in vivo validation of the HIF-1α/ACOX1/HSP90AB1 K265cr/TXN axis in OSCC. (A) Western blot analysis of the indicated proteins in 24 human OSCC tumor tissues. (B to F) Scatter plots of band intensity measurements from panel (A): (B) HIF-1α and TXN, (C) HIF-1α and HSP90AB1 K265cr, (D) HSP90AB1 K265cr and TXN, (E) ACOX1 and HSP90AB1 K265cr, and (F) HIF-1α and ACOX1 ( n = 24, Pearson correlation). (G) TXN protein levels in 2 OSCC patient groups receiving postoperative chemoradiotherapy ( n = 152; **** P < 0.0001, unpaired t test). (H) Schematic diagram of the cisplatin-treated xenograft experimental design (ip, intraperitoneal injection). (I) Body weight measurements of the mice during the treatment period (ns, not significant, unpaired t test). (J) Western blot analysis of the indicated proteins in xenograft tumor tissues. (K to O) Scatter plots of band intensity measurements from panel (J): (K) HIF-1α and TXN, (L) HIF-1α and HSP90AB1 K265cr, (M) HSP90AB1 K265cr and TXN, (N) ACOX1 and HSP90AB1 K265cr, and (O) HIF-1α and ACOX1 ( n = 14, Pearson correlation).

Article Snippet: The His-tagged HSP90AB1 fusion protein and His peptide were purchased from Cusabio Technology Co., Ltd. (Wuhan, China) and Yeasen Biotechnology Co., Ltd. (Shanghai, China).

Techniques: In Vivo, Biomarker Discovery, Western Blot, Injection

Journal: Research

Article Title: Hypoxic Reprogramming of ACOX1-Driven HSP90AB1 Crotonylation Stabilizes Thioredoxin to Orchestrate Redox Homeostasis in Oral Squamous Cell Carcinoma

doi: 10.34133/research.1129

Figure Lengend Snippet: The schematic diagram of the HIF-1α/ACOX1/HSP90AB1 K265cr/TXN signaling axis in OSCC.

Article Snippet: The His-tagged HSP90AB1 fusion protein and His peptide were purchased from Cusabio Technology Co., Ltd. (Wuhan, China) and Yeasen Biotechnology Co., Ltd. (Shanghai, China).

Techniques: